Immunoblot Analysis of Protein Expression

For immunoblots, equal total protein of cell lysates (2.5–20 μg) were detected with antibodies to mouse p53 (FL393), human p53 (PAb1801; Santa Cruz Biotechnology), HSF1, p-Ser326 HSF1, AKT, pAKT, Erk, pErk, Hsp70, Hsp90, Hsp90α, EGFR, EGRF-Tyr845P (all Cell Signaling, Danvers, MA, USA), ErbB2, actin, GAPDH, GTS, GFP, HSc70 and HDAC1 (all Neomarkers, Fremont, CA, USA).^{35 (link), 36 (link), 37 (link)} SDS-PAAG gels (6%) were used to detect slower migrating HS-activated HSF1 used in experiments presented in Figures 2 and 3. SDS PAAG (10%) gels were used in all other experiments. Co-immunoprecipitations were performed with 1 μg of antibody overnight. Beads were washed with SNNTE and RIPA buffers 3 × each, followed by blotting.

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Li D., Yallowitz A., Ozog L, & Marchenko N. (2014). A gain-of-function mutant p53–HSF1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress. Cell Death & Disease, 5(4), e1194-.

Publication 2014

PAb1801 pAKT pErk Hsp70 Hsp90 Hsp90α EGRF-Tyr845P

Actin Antibodies Antibody Buffers Cell Co immunoprecipitations Egfr Erbb2 Gapdh Gels Hsp70 Hsp90 Human Immunoblots Mouse Paag Protein Ripa

Corresponding Organization : Stony Brook University

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Total protein of cell lysates (2.5–20 μg)

dependent variables

Detection of antibodies to mouse p53 (FL393)
Detection of antibodies to human p53 (PAb1801; Santa Cruz Biotechnology)
Detection of antibodies to HSF1
Detection of antibodies to p-Ser326 HSF1
Detection of antibodies to AKT
Detection of antibodies to pAKT
Detection of antibodies to Erk
Detection of antibodies to pErk
Detection of antibodies to Hsp70
Detection of antibodies to Hsp90
Detection of antibodies to Hsp90α
Detection of antibodies to EGFR
Detection of antibodies to EGRF-Tyr845P
Detection of antibodies to ErbB2
Detection of antibodies to actin
Detection of antibodies to GAPDH
Detection of antibodies to GTS
Detection of antibodies to GFP
Detection of antibodies to HSc70
Detection of antibodies to HDAC1

control variables

SDS-PAAG gels (6%) used to detect slower migrating HS-activated HSF1 used in experiments presented in Figures 2 and 3
SDS PAAG (10%) gels used in all other experiments
Co-immunoprecipitations performed with 1 μg of antibody overnight
Beads washed with SNNTE and RIPA buffers 3 × each, followed by blotting

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